The overall objective is to develop and implement a High Throughput Screen suitable for identifying compounds with selectivity for nicotinic acetylcholine receptors which contain the alpha6 subunit (alpha6 nAChRs). To meet this objective, two Specific Aims are proposed: 1. An HTS-ready assay will be established and optimized for an existing, highly-functional, monoclonal cell line stably transfected with alpha6/Beta2Beta3 nAChRs. Both membrane-potential and intracellular Ca2+ dye approaches are suitable, and the approach which can be refined to provide the most robust and reliable results will be adopted for the second Aim. 2. The optimized assay will be configured for HTS conditions. An overall screening workflow is proposed. This workflow incorporates secondary orthogonal- potency-, and selectivity-counter-screening protocols to identify and discard false positives, and to characterize confirmed candidates, from the initial screen. Already-available assays suitable for orthogonal- and counter-screening, using different activities than the primary screen, are described. The second Aim also contains a proposal to generate proof-of-principle data from a pilot screen of ~2400 structurally-diverse test compounds (including a high proportion of established CNS-active drugs). The health relevance of this project arises from genetic association and neurochemical studies which link alpha6 nAChR subunit gene variants and alpa6 nAChR function to substance use and dependence (particularly to tobacco smoking), and to important features of Parkinson's Disease development and treatment. Each of these conditions is a major public-health issue. The underlying molecular mechanisms by which alpha6 nAChRs influence these phenomena are still not well understood. Compounds identified by the proposed screen could become valuable research tools to understand better the etiology of the major public health issues associated with alpha6 nAChRs. This would provide impact through new scientific insights and potentially by revealing novel therapeutic avenues / targets. Compounds identified by this screen could also be useful treatments for the same conditions. Notably, the impact on drug dependence therapies could be quite broad, given the established association of alpha6 nAChRs with abuse liability for diverse substances. Further, an alpha6 nAChR- selective radiotracer could be a valuable PET/SPECT ligand for Parkinson's Disease diagnosis and/or monitoring of treatment success. PUBLIC HEALTH RELEVANCE: This project is intended to develop and implement a technique for rapidly testing a large library of compounds, with the objective of identifying and characterizing compounds selective for nicotinic acetylcholine receptors which contain the 16 subunit. These receptors have been associated with the following major public- health issues: smoking, alcohol abuse, and Parkinson's Disease. Compounds identified by this project would be useful in understanding the underlying causes of these conditions, and may serve as leads for the development of compounds used in their treatment and / or diagnosis.